Combinations of Tibetan tea and medicine food homology herbs: A new strategy for obesity prevention

Abstract Obesity has become a significant global public health problem. Functional drinks have been an essential direction for obesity prevention research. The present study investigated the preventive effect and safety of winter melon and lotus leaf Tibetan tea (WLTT, a compound tea drink based on Ya'an Tibetan Tea and medicine food homology herbs) on obesity. The rats' hypercaloric high‐fat diet (HFD) obesity model was established to evaluate obesity prevention and explored the mechanism through intestinal flora regulation. The results showed that in obese rats with the intervention of WLTT (400, 800, and 1600 mg/kg BW), the body weight, fat accumulation, adipocyte cell size, serum lipid levels, and antioxidant enzyme activity (SOD, GSH‐Px, and MDA) were progressively improved. 16S rRNA high‐throughput sequencing showed that WLTT could improve intestinal flora disorders due to HFD, which significantly reversed the relative abundance of Firmicutes and the F/B ratio associated with an HFD, and significantly upregulated the relative abundance of Verrucomicrobia. At the genus level, the downregulation of the relative abundance of Akkermansia and unclassified_Lachnospiraceae groups, and the upregulation of the relative abundance of Romboutsia, Ruminococcus, Corynebacteriume, and Saccharibacteria_genera_incertae_sedis groups brought about by the HFD were significantly reversed. The results of the above experiments were compared favorably with those of a parallel experiment with Bi ‐Sheng ‐Yuan slimming tea (BSY, a functional drink based on green tea and medicine food homology herbs). Overall, the findings have provided that WLTT can prevent obesity owing to an HFD by regulating intestinal flora and has a good safety profile, and combinations of Tibetan tea and medicine food homology herbs could be a new option for obesity prevention.


| INTRODUC TI ON
With economic development, rich diets, and changes in lifestyle habits, the prevalence of obesity increases annually. According to the latest national prevalence estimates for 2015-2019, 6.8% were overweight and 3.6% obese in children under 6 years old, 11.1% overweight, and 7.9% obese in children and adolescents aged 6-17 years old, 34.3% overweight and 16.4% obese in adults (≥18 years), and with a population base of 1.41 billion, the obesity situation in China is no longer optimistic (NCD Risk Factor Collaboration [NCD-RisC], 2016; Pan et al., 2021). Many studies have shown that obesity is a high-risk factor for many chronic diseases such as diabetes, hypertension, hyperlipidemia, and coronary heart disease, and is also a paramount factor in increasing cerebrovascular disease, increasing the load on the heart, causing bone and joint disease, and predisposing to cancer (Banerjee et al., 2020;Marini et al., 2020;Oliveira et al., 2020;Seravalle & Grassi, 2017).
Improving obesity and losing weight has become a long-standing wish of obese patients worldwide. However, because there is no specific medicine for obesity, the lack of long-term self-discipline required for diet control and physical exercise is why most obese patients fail to lose weight (Carbone et al., 2019). Thus, the search for and development of food-grade safe and healthy beverages for long-term prevention may be an essential step toward improving the situation described above.
Tea is known as one of the safest and healthiest beverages globally and is consumed by people worldwide (Chen et al., 2015;Chung et al., 2020;Li et al., 2019;Liu et al., 2022;Shen et al., 2019;Vieux et al., 2019). Ya'an Tibetan Tea is a classic tea variety fermented in the Ya'an region of Sichuan Province in China with a unique geographical environment with exquisite traditional techniques and has a history of over 1300 years (Chen, 2017).
Numerous studies have confirmed that Ya'an Tibetan Tea has a wide range of biological activities in terms of antioxidants, regulation of obesity, and related metabolic syndrome. (Liu et al., 2022;Xie et al., 2018;Zheng et al., 2020;Yuan et al., 2016). In order to enrich the flavor and taste of Ya'an Tibetan Tea and enhance its functionality, the research team has developed WLTT based on Ya'an Tibetan Tea, using the traditional Chinese medicine compounding concept and incorporating medicine food homology herbs. In order to explore the modulating effect of WLTT on obesity and its potential mechanism, the group studied the intervention effect of WLTT on obesity in a highcalorie model of obese rats and evaluated its safety, and analyzed its potential mechanism through the regulation of intestinal flora disorder. At the same time, the team conducted a comprehensive and systematic comparative study of BSY, which is mainly green tea and herbal medicine with a large customer base in China, to fully explore the feasibility of combining Tibetan tea and medicine food homology herbs.  Co., Ltd. (Lot No. 19201102). Superoxide dismutase (SOD), malondialdehyde (MDA), and glutathione peroxidase (GSH-Px) kits were purchased from Nanjing Jiancheng Institute of Biological Engineering.
Chloral hydrate and formaldehyde were all analytically pure and purchased from Sinopharm Chemical Reagent Co., Ltd.

| Preparation of WLTT and BSY
Winter Melon and Lotus Leaf Tibetan Tea was converted to a medium dose of 800 mg/kg in experimental rats based on the recommended daily human dose of 8 g and then designed for a low dose of 400 mg/kg and a high dose of 1600 mg/kg. WLTT was decocted for 20 min at a tea-to-water ratio of 1:40 for the first decoction and for 10 min at a tea-to-water ratio of 1:20 for the second decoction to obtain the tea broth, which was concentrated to the corresponding raw tea concentration for both decoctions based on a gavage volume of 10 ml/kg for rats. BSY was converted to an experimental dose of 500 mg/kg in rats based on the recommended daily human dose of 5 g. The decoction method is the same as for WLTT and is also concentrated to the corresponding raw tea concentration based on the rats' gavage volume. The above samples were decocted once every day, concentrated, and then refrigerated at 4°C. According to the method for functional evaluation of healthy food, 70 SPF-rated SD male rats were taken and fed for 1 week after acclimatization. The rats were randomly divided into two groups according to body weight, 10 of which were normal control (NC) and given maintenance chow, and the remaining 60 were a hypercaloric high-fat diet model group and given hypercaloric chow. After 2 weeks of feeding, 60 rats given hypercaloric chow were sorted by weight gain, and after eliminating the obese-resistant rats with lower weight gain, the screened 40 obese-sensitive rats were again randomly allocated into five experimental groups with 8 rats each according to their body weight range. They were hypercaloric high-fat diet model group (HFD), Bi-Sheng-Yuan slimming tea 500 mg/kg (HFD + BSY), WLTT 400 mg/kg group (HFD + L-WLTT), WLTT 800 mg/kg group (HFD + M-WLTT), and WLTT 1600 mg/kg group (HFD + H-WLTT).

| Animal experimental protocol
Each experimental group was given the corresponding concentration of the test sample, and the NC group and the model group were given double-distilled water as the control. All rats were oral gavage at a dose of 10 ml/kg once a day, and the dose was adjusted by weighing once a week. All animal experiments were conducted with the Ethics Committee of Zunyi Medical University (approval no: ZMUER2020-1-057).

| Sample collection and biochemical index and histological analysis
At the end of the intervention cycle, the rats were fasted without water for 12 h, weighed, anesthetized with 10% chloral hydrate by intraperitoneal injection, and collected blood from the femoral artery. After the plasma was left to stand for 2 h, the serum was separated by centrifugation at 3000 r/min at 4°C for 10 min, and taking a partial serum sample was quickly sent to the Laboratory

| 16S rRNA high-throughput sequencing of gut microbiota of cecum
The cecum contents of three rats were randomly collected from each group and sent to Sangon Biotech (Shanghai) Co., Ltd. for highthroughput sequencing library construction and Illumina Mi Seq sequencing. The raw data obtained based on sequencing were stitched according to overlap relationships. The samples were differentiated and then quality-controlled and filtered for sequence quality, followed by OTU clustering and species annotation, Alpha diversity analysis, Beta diversity analysis, grouping test analysis, and species taxonomic composition analysis.

| Statistical analysis
The experimental data were statistically analyzed using GraphPad Prism 8.0 software (GraphPad Prism Software). One-way analysis of variance was used for comparison between groups, and data are expressed as X ± SD. Differences of p < .05 were considered statistically significant.

| Effect of WLTT and BSY on body weight, food intake, Lee's index and fat cells
After 2 weeks on a hypercaloric diet, rats showed significant weight differentiation, thus demonstrating the model's success. However, with the intervention of the experimental samples, the weight of each experimental rat further diverged. The weight-loss trend was significantly better in each experimental group of WLTT than in the BSY group, showing a dose-dependent trend (p < .05, Figure 1a).
However, the results of monitoring the change in food intake of each experimental group weekly showed that after the experimental groups were fed high-fat chow, there was a tendency for the food intake of each group to decrease. Luckily, there was no difference between each sample intervention group and the HFD group, and the food intake curve fit (Figure 1b). In addition, statistical analysis of the weight gain in each group for the experimental cycle showed similar results (p < .05, Figure 1c). Analysis of Lee's index in rats revealed that both the WLTT and BSY groups down-regulated Lee's index in obese rats (p < .05, Figure 1d).

| Effect of WLTT on liver and kidney function
The safety of WLTT was also of great interest to the group. The results showed that WLTT significantly downregulated the liver index and ALT in obese rats (p < .05, Figure 2a

| Effect of WLTT and BSY on the intestinal flora
Analysis of the 16S rRNA gene sequencing showed that the sequencing depth was primarily sufficient to cover the samples' strain information. The Rank-abundance curve is wide and flat, indicating a good diversity of intestinal microflora (Figure 3a). Beta diversity analysis of species principal component analysis analyzes the more similar the species composition of the sample (Figure 3b). The subgroup tests' partial least squares discriminant analysis showed differences in microbial communities between groups (Figure 3c).  Note: Data were expressed as a mean ± SD, n  Figure 4c). This result also clearly suggests that WLTT at the phylum level has the potential to reverse the intestinal flora disorders associated with the hypercaloric model. Meanwhile, BSY also showed some ability to improve intestinal flora disorders, but the results in the Proteobacteria were not as good, with a statistically significant upregulation compared to the model group (p < .05, Figure 4c).  Figure 4f) and Ruminococcus (p < .05, Figure 4f). The BSY group also showed some intervention in the above flora. However, the overall evaluation was not as good as that of the HFD + H-WTLL group, especially in the relative abundance of Psychrobacter (p < .01, Figure 4f) and Aerococcus (p < .05, Figure 4f) showed a significant upregulation trend. However, the WTLL experimental groups did not show statistical differences (Figure 4f).
In order to explore the effect of the WLTT on the regulation of intestinal flora and thus potential functions, the team compared the species composition obtained from sequencing with the KEGG PATHWAY database to infer the composition of functional genes in the samples. STAMP differences between the WLTT intervention groups and the HFD group were analyzed by Welch's t-test and found that the WLTT interventions resulted in altered functional microbial abundance compared to the HFD group and that the number of statistically different functional metabolic pathways increased progressively with increasing dose. It mainly causes changes in some metabolic pathways, such as amino acid, insulin, and bile metabolism, altering the biosynthesis and degradation of some secondary metabolites ( Figure 5).

| DISCUSS ION
The worsening trend of global obesity cases has been regarded as the epidemic of the 21st century, and medical and nutritional experts around the world are working hard to address this problem.
The development of functional drinks from natural foods that intervene in obesity has been an important direction in obesity Akkermansia muciniphila, a normal flora of the human intestine, is the best-known probiotic in Akkermansia, and numerous studies have shown a negative association between Akkermansia muciniphila and diseases such as obesity, diabetes, cardiovascular disease, and low-grade inflammation (Higarza et al., 2021;Zhang et al., 2021).
Lachnospiraceae has also been shown to reduce obesity and improve inflammation as a potentially beneficial bacterium (Naudhani et al., 2021;Truax et al., 2018). The group found that WLTT and BSY significantly increased the relative abundance of Akkermansia, un-classified_Lachnospiraceae at the genus level, suggesting that WLTT F I G U R E 5 The effect of the function of microbial communities predicted by PICRUST. The left-hand side of each graph shows the proportion of abundance of different functional categories in the two samples, the middle shows the proportion of differences in functional abundance within the 95% confidence interval, and the far right-hand side shows the p-value, with p < .05 indicating a significant difference, and is marked in red. Aerococcus was confirmed as a human pathogen in numerous studies (Rasmussen, 2016;Tai et al., 2021), which led the group to conclude that there is some risk potential in the regulation of flora by BSY.
The group compared the species composition obtained from sequencing with the KEGG PATHWAY database and found that the intervention of the WLTT groups led to changes in the functional abundance of microorganisms, and the number of statistically different functional metabolic pathways increased with increasing dose. It mainly causes changes in some metabolic pathways, such as amino acid, insulin, and bile metabolism, in addition to altering the biosynthesis and degradation of some secondary metabolites, which are mostly positively associated with obesity and lipid metabolism.

| CON CLUS IONS
In summary, WLTT has a significant preventive effect on a highcalorie model of obesity in rats, while the drink also has a good safety profile. The regulation of the intestinal flora significantly, the increase in the abundance of Akkermansia and unclassified_ Lachnospiraceae, and the decreases in the abundance of Romboutsia and Ruminococcuse, thus affecting changes in metabolic pathways such as amino acid, insulin, and bile metabolism and the biosynthesis and degradation of secondary metabolites, may be the key reasons for the effectiveness of WLTT. The combination of Tibetan Tea and medicine food homology herbs may be superior to green tea and medicine food homology herbs in terms of function and safety, which could be a new strategy for obesity prevention.

CO N FLI C T O F I NTE R E S T
The authors declare no conflicts of interest.